The phytochemical analysis was carried out for standardization of fruits of Opuntia elatior Mill. The fruits were preliminary evaluated by estimation of proximate analysis. Presence of betacyanin was confirmed by spectrophotometric and high performance liquid chromatography coupled with mass spectroscopy techniques. The average weight of fruit was 24.568 ± 7.134 g/unit and the percentage of peel and seed was very low compared to the edible portion. Phytochemical analysis indicated the presence of color pigment betacyanin as an active principle and sugar content in high amount and low acidity of fruit which make it very sweet and delicious. The total betacyanin content (47.10 mg/100 ml) equivalent to betanin obtained from fruits of Opuntia elatior Mill. was higher as compared to Opuntia ficus-indica and Opuntia undulata Griff. while lower as compared to Opuntia stricta Haw.